The present invention relates generally to the field of reading X-rays, electrophoresis gel bands, and the like wherein the size and darkness of an image provides useful information. More specifically the invention has to do with a method of quantitating images in X-ray film and electrophoresis gel bands utilizing a spectrophotometer to quantify the silver which comprises the image. Presently the conventional method of quantitating such images is by densitometry, a method which can be prohibitively expensive and impractical.
Often in various professions, such as biochemistry, molecular biology, cell biology, dentistry and medicine, X-ray films are developed in which images represent a chemical or tissue. The darkness of the image is proportional to the amount of material present. It is frequently desirable to quantitate the material by judging the darkness or size of the image. Until now, the main way for quantitating images on X-ray film is to use an instrument called a densitometer.
A densitometer can quantitate optical densities, such as those of bands or spots on gels, membranes and autoradiograms. Densitometry can be used in order to create digital records of these samples. Densitometers work by comparing the intensity of light transmitted through a sample with the intensity of light incident on the sample. Thus, densitometers are instruments that are specially designed and manufactured for image quantitation.
For example, the ImageMaster.TM. Electrophoresis Evaluation System, manufactured by Pharmacia LKB Biotechnology, is available for protein quantification and analysis, and compared to other densitometry instrumentation, purports to be faster and better equipped to scan more types of images. The ImageMaster.TM. System DTS, has various uses, for instance, scanning wet or dry gels, autoradiographs, blots, photographs, slides, 96-well microtiter plates and Petri dishes. The system comprises a high resolution, white light, transmittance/reflectance desk top scanner (DTS) and computer software. However, the sole use of the spectrophotometer in the present invention eliminates the need for all of these expensive components.
Moreover, the use of a densitometer has two disadvantages when compared with the present invention. The first disadvantage is the cost and specialized use of the densitometer. It is only used for quantitating images, which makes it prohibitively expensive for the occasional user or for laboratories lacking money to buy instruments. The second disadvantage is that a densitometer may be designed only to accept certain sizes and shapes of X-ray film. Until now, no simple inexpensive physical or chemical method existed for quantitating images on X-ray film. The present invention was developed in order to circumvent these problems.
The present invention addresses the problems in the prior art by utilizing a spectrophotometer, an instrument readily available in research and clinical laboratories. The basis for the use of this instrument is the finding that silver, isolated from various sources of images, when bound with a complexing agent, will produce a color change that can be read with the spectrophotometer. The complexing agent, specifically, diphenylthiocarbazone (dithizone) has been known in the art to change colors when complexing with various metals especially in the context of the elimination of adventitious metals. Holmquist, B. (1988) Methods Enzymol. 158, 6-14. However, in the present invention, we have discovered that the complex formed between silver and the dithizone complexing agent has high affinity and a different color than free dithizone thus, making the extent of the color change linearly proportional to the amount of silver added to the dithizone solution. Therefore, upon comparison of the amount of silver in the silver containing image with the amount of silver in a reference spot or a standard curve, the amount of material is readily quantifiable.
Because silver compounds comprise photographic images, one application of this invention is for use in quantitating X-ray film images that represent a given material. When a photographic material (containing silver bromide) is subjected to prolonged exposure, a reaction will take place to such an extent that the photo-reacted silver causes a visible darkening. D. H. O. John & G. T. J. Field, A Textbook of Photographic Chemistry, London, Chapman & Hall Ltd., 1963. It is the silver from these darkened photographic film images which can be isolated and then quantitated by the spectrophotometric procedure.
An additional application of this invention is in quantitating the silver in bands in silver stained electrophoresis gels. By using the same method as used with X-ray films, a linear correlation is found between the amount of material in a given gel band and the amount of absorbance resulting from dithizone-silver complex formations. Since there is a need for an inexpensive and practical quantitation procedure, this method presents a convenient, inexpensive alternative to using a densitometer for quantitating images.
It is therefore a principal object of the invention to provide for a more economical method of quantitating the silver in silver stained bodies without the need for the highly specialized and expensive densitometer. Densitometers are relatively rare and expensive, often ranging from $15,000 to $30,000, whereas spectrophotometers are often comparatively inexpensive. Because the present invention utilizes a spectrophotometer, an instrument common to most laboratories, the process is convenient, rapid and inexpensive. Moreover, unlike a densitometer which is very limited in use because of its specialized purpose, a spectrophotometer has a multitude of uses beyond the applications of the present invention.
It is a further object of the invention to provide improved methods of determining relative amounts of silver in X-ray film images generated, for example, by autoradiography or chemiluminescence. Until now, the most common method of quantitating images in X-ray film was by using a densitometer. But, a densitometer is highly specialized and consequently limited in use in that it may be designed to accept only certain sizes and shapes of X-ray film. These limiting features make it prohibitively expensive for the casual user. The present invention overcomes these problems because direct quantitation of the X-ray image is achieved regardless of the size and shape of the X-ray film. Since the present invention requires the use of a spectrophotometer, a common fixture in biological and clinical laboratories, it provides a convenient, inexpensive alternative to using a densitometer. Thus. images on X-ray films can be quantitated in terms of the amount of silver that is present in the images.
It is yet a further object of the invention to provide improved methods of determining relative amounts of material in silver stained electrophoresis gels. Experiments with silver stained gels demonstrate that the amount of quantitated silver in a band in the gel relates to the amount of material represented by the band. Of course, this invention teaches a general method which contemplates many diverse applications. It may not only be applied in the fields such as biology and chemistry but can extend into many other fields, such as metallurgy and jewelry making, where it may be necessary to quantitate silver.